Unsaturated membrane lipids in cells confronted with physical or chemical oxidative insults may undergo peroxidative damage, often with deleterious consequences. Lipid hydroperoxides (LOOHs), including phospholipid (PL)- and cholesterol (Ch)-derived species, are important non-radical intermediates in such reactions. Once formed, LOOHs can have a variety of fates which impact on the viability of a targeted cell. A nascent LOOH may undergo iron-catalyzed one-electron reduction to free radical species, which trigger damaging (toxicity-enhancing) chain peroxidation reactions. Alternatively, selenoperoxidase (GPX4)-catalyzed detoxification of LOOHs may occur, i.e. two-electron reduction to relatively innocuous alcohols. While studying these processes, we have identified a new pathway, which expands the LOOH dynamic, viz. intermembrane translocation. The proposed research will delve more deeply into this phenomenon with two hypotheses in mind: (i) LOOHs move between membranes more rapidly than parent lipids, both intra- and intercellularly, and this is enhanced by transfer proteins. (ii) Availability of redox iron or GPX4 at acceptor sites will determine whether translocation expands or attenuates LOOH cytotoxicity. The studies will utilize GPX4-null COH-BR1 breast tumor cells, transfectants thereof overexpressing either mitochondrial or non-mitochondrial GPX4, and transfectants overexpressing non-specific lipid transfer protein (SCP-2). High sensitivity/specificity techniques for detecting LOOHs and other oxidation products will be employed, viz. HPLC-EC(Hg) and HPTLC-PI, both of which were developed in this laboratory. The hypotheses will be tested by studying (i) spontaneous and SCP-2-facilitated LOOH transfer between plasma membranes (PM) and mitochondria (Mito) isolated from tumor cells; (ii) one-electron vs. two-electron reactivity of transfer-acquired LOOHs in PM and Mito; (iii) effects of SCP-2 overexpression on subcellular LOOH trafficking and cytotoxicity; and (iv) possible cooperative effects of SCP-2 and GPX4 co-overexpression on LOOH detoxification. By exploring this new dimension in LOOH bioreactivity, these studies will provide valuable new insights into the cytopathological effects of these species.